Synergy among biologically active agents is a well known phenomenon. Usually the synergy is discovered either serendipitously or as a result of deduction that synergy should take place from understanding of the biological process. For example, combination of penicillin with an inhibitor of penicillinase (the enzyme that degrades penicillin) would be expected to yield a higher performing antibiotic—which indeed it does.
As scientific understanding of the complexity of living processes has increased, it has become clear that the extent to which any single biological end point is affected by many different pathways is much greater than had been expected. In particular, it has become clear that mammalian and other cells have enormous redundancy within their pathways. Nowhere has this been clearer than in the results of gene knockout experiments where the complete deletion of a gene thought to have a critical function often results in no detectable effect on the organism.
A consequence of this complexity is that many desirable biological end effects might be most easily achieved by intervening simultaneously in several pathways, and/or at multiple points within such pathways, rather than focusing on finding a single highly potent agent to act at one vital “node” within the network of pathways.
A particularly clear way to understand this phenomenon is to consider the common concept of the “rate-limiting step”. Conventionally, in biology, it is believed that one step within a pathway is usually rate limiting, i.e., inhibition of that step will have immediate effect on the whole pathway while inhibition of other steps will have little or no effect. Such rate limiting steps are thus the normal target for development of drugs. Consider however what happens in a complex, highly interlinked, system of pathways. When the rate-limiting step is inhibited by an active agent, other pathways within the system compensate for that inhibition. In this new “compensated” configuration, different pathways may become rate limiting and far greater impact on the overall system will be achieved by adding an agent that acts on this new rate limiting step than would be achieved by further inhibiting the, original rate limiting step. The same logic applies when one adds a third agent to the mixture—it is likely that yet a third pathway will have become rate limiting when both of the initial rate limiting pathways have been inhibited.
This paradigm of “multiple active intervention” as source of synergy is of relevance wherever discovery of biologically effective agents is desired, including in the pharmaceutical industry. The approach is however of particular interest in the industry sectors such as cosmetics, personal care products or dietary supplements where regulatory rules do not make use of complex mixtures of active agents difficult. Of particular importance in these sectors is the potential that combinations of active agents that individually are of low potency and broad specificity might in combination be highly potent and highly selective. This property of a combination to be more potent and/or selective than expected based on the individual components is defined here as “synergy” and the mixture of ingredients displaying this synergy is what is meant as a “synergistic” mixture.
Finding such highly efficacious combinations, i.e., synergistic mixtures, of active agents is challenging however. Serendipity is not a valid route as the number of potential combinations of agents is staggeringly large. For example, there are trillions of possible 5 fold combinations of even a relatively small palette of 5000 potential agents. The other normal discovery strategy of deducing potential combinations from knowledge of mechanism is also limited in its potential for the following reasons.
Many biological end points of living organisms are affected by multiple pathways. These pathways are often not known, and even when they are, the ways in which the pathways interact to produce the biological end effect are often unknown. By biological end effect is meant the ultimate biological effect that is desired of the agent or combination, e.g., hair growth stimulation or the slowing or reversal of the skin ageing process. To further complicate the picture, many potential active agents have broad specificity (in pharmaceutical parlance they are “dirty drugs”—like aspirin) and therefore simultaneously affect multiple pathways. The mathematical tools and data banks needed to model and thus understand such complexity, do not yet exist and so use of “mechanism based” discovery of multiple synergies can rarely be achieved.
A useful but limited approach to finding highly synergistic combinations of agents was described in WO 02/02074. In this case a number of specific steps were identified within the retinol metabolism pathway, which were predicted on theoretical grounds to act synergistically with each other. Actives effective on each step were identified through highly specific assays such as enzyme inhibition and the resulting smaller set of effective compounds were tested in high order combinations. High levels of synergy were found. This approach however is limited to the special case where the pathway of interest is well understood and can thus be theoretically modeled.
There thus remains a pressing need for effective methods capable of discovering these multiple synergies, particularly those involving multiple unknown or poorly understood pathways, in a timely and cost effective manner.
A particularly significant advantage of the experimental strategy and methods disclosed herein is the increased likelihood of identifying highly synergistic combinations of biologically active materials without requiring impractically large numbers of experimental tests.
An additional advantage especially when used with the assay methods of the invention is the ability to utilize high throughput experiment methods in biological models that incorporate the multi-pathway features of real systems of interest.
A still further advantage of the strategy and methods is in the identification of highly synergistic combinations that will be suitable for use by humans.
These and other advantages will become clear from the description of the invention.
The following additional patents and publications have been considered:
Systematic discovery of multicomponent therapeutics by Alexis A. Borisy, Peter J. Elliott, Nicole W. Hurst, Margaret S. Lee, Joseph Leha'r, E. Roydon Price, George Serbedzija, Grant R. Zimmermann, Michael A. Foley, Brent R. Stockwell, and Curtis T. Keith. Proceedings of New York Academy of Science, Jun. 24, 2003, Vol. 100, part 13, pp 7977-7982. This publication describes the principles of why unpredictable biological synergy among agents is expected to occur and demonstrates the detection of binary synergies among libraries of potential drugs using a conventional method of testing all possible binary synergies between drugs in the library. 36 tests are required for each binary combination and extremely simple and ultra high throughput assays are developed to make possible the enormous number of assays needed to probe merely binary synergies within the library. No suggestion is made that more experimentally efficient strategies to discover synergies are possible and the method is clearly impractical should 3 fold or higher orders of synergy be sought because of the large number of experiments required.
GB 2242978 (Phillpot et al) describes a method for assessing materials for hair growth or pigmentation activity whereby individually microdissected hair follicles are grown in culture medium. This method is the standard method used by almost all research groups needing an in vitro hair growth assay (the patent having been abandoned). The method is however fundamentally unsuited to any high throughput application due to the laborious process of microdissecting hair follicles.
None of the references cited above teaches the specific methods to assess biologically active materials and to identify synergistic combinations as disclosed herein. The inventor is not aware of any prior art that teaches the experimental design strategy or assays disclosed herein.